The development of oligonucleotide-directed mutagenesis is perhaps the most influential tool in the study of protein structure and function. Existing methods include “Quikchange” mutagenesis, the Kunkel method, and enzymatic inverse PCR. As they are all based on annealing synthetic DNA, every desired mutant construction involves a pair of mutagenic oligonucleotides, a thermocycling reaction, and subsequent cloning and sequence verification of the mutated genes. This places both manpower and financial limitations on high-throughput mutagenesis studies such as alanine scanning and directed evolution. Practicality dictates that it is nearly impossible to efficiently make every alanine, or other amino acid, mutation of a 500 amino acid protein using current methods. Accordingly, there is a need for new mutagenic techniques that offer greater efficiency and control.